Endospores are produced by very few types of bacteria, most notably the genera Clostridium and Bacillus. These protective structures are made through a process known as sporulation in response to extreme environmental conditions, such as high temperatures, desiccation, chemicals, changes in pH and lack of food.

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SCIENCE PHOTOS

Photographic Guide to Completing an Endospore Stain

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The procedure for differentially staining endospores and vegetative cells is as follows:

Place the heat-fixed bacterial slide over screened water bath and then apply the primary stain malachite green.

Allow the slide to sit over the steaming water bath for 5 minutes, reapplying stain if it begins to dry out.

Remove the slide from the water bath and rinse the slide with water until water runs clear.

Application of Primary Stain: 1. Endospore stain set-up; 2 & 3. Malachite green being applied to slides on water bath, note in photo 3, clothes pins are used to make handling the slide easier; 4. Rinse. Make sure to rinse thoroughly enough so that there are no "chunks" of green on slide.

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After this staining procedure, the endospores will appear green, having retained the primary stain, malachite green. Vegetative cells (bacteria are in the active, metabolizing state) will appear pink, having retained the counterstain, safranin.

BACTERIAL SMEAR AND ENDOSPORE STAIN VIDEOS

How to Do an Endospore Stain

In the dormant, inert endospore state, bacteria do not metabolize or reproduce, but exist in a type of suspended animation, much like the seeds of plants do. When environmental conditions again become favorable, the endospore germinates, resulting in a new vegetative cell.

Preparing a Bacterial Sample

Prior to staining bacteria, a bacterial smear must be heat fixed onto a microscope slide. A smear is a sample of bacteria suspended in a small amount of water on a slide. That sample is then dried using heat. The heat kills the bacteria and attaches the sample to the slide so that it does not easily wash away.

Staining Bacterial Endospores

Normal water-based techniques, such as the Gram stain, will not stain these tough, resistant structures. In order to stain endspores, the dye malachite green must be forced into the spore with heat, in much the same way that carbol fuschsin is forced through the waxy mycolic acid layer of Mycobacterium in the Acid-fast stain.